The broad long-term goals of this research are to elucidate the mechanisms regulating uracil-DNA repair in mammalian cells. Uracil-DNA glycosylase (UDG) is a critical DNA repair enzyme that functions in the correction of transition mutations caused by cytosine deamination. In mammalian cells two forms of this highly conserved enzyme exist, one that is directed to the mitochondria (UDG1) and one that is directed to the nucleus (UDG1A). Significant information exists on the structural and biochemical properties of the mitochondrial form of this enzyme. In contrast, very little information exists on the nuclear form of this enzyme. In cells, UDG1A is post-translationally modified by phosphorylation at serine and threonine residues. Preliminary data demonstrate that at least one of these sites is phosphorylated in vitro by a cyclin dependent kinase. Phosphorylation, by the cyclin-dependent kinase increases the activity of this DNA repair enzyme. This proposal specifically addresses the following key issues. Does the phosphorylation pattern of nuclear UDG1A change with the phases of the cell cycle or under conditions of elevated dUMP incorporation into DNA (aim 1)? Is the cyclin-dependent kinase consensus site of the nuclear form of UDG phosphorylated in vivo? What are the other phosphoamino acid residues of nuclear UDG1A (aim2)? Does phosphorylation at the cyclin-dependent kinase site of UDG1A, alone, control enzymatic activity? Do other phosphorylation contribute to regulating UDG1A enzymatic activity (aim 3)? Does phosphorylation of UDG1A instigate association with other proteins of the cell (aim 4)? Utilizing a cell culture model we will determine cell-cycle phase specific phosphorylation and determine if there is DNA damage-induced changes in the phosphorylation pattern of UDG1A. Using biochemical and recombinant DNA technologies we will establish which amino acid residues of UDG1A are phosphorylated and which are responsible for modulating activity. We will also determine if UDG1A is in association with other proteins of the cell. The overall experimental goal of this proposal is to gain a better understanding of this post-translational mechanism. Our data indicate another level of regulation of this DNA repair enzyme. Since UDG1A is the first step in this DNA repair pathway, regulation of UDG1A activity will dictate control over the enitre pathway.